The ability to routinely synthesize ribonucleic acid (RNA) has become increasingly important as research reveals the multitude of RNA's biological functions. There are many types of RNA including ribosomal RNA, transfer RNA and messenger RNA. RNA also is important in various structures and functions as well as being a catalyst in enzymatic reactions, as in the case of ribozymes. Because of the important biological roles RNA plays, both known and unknown, it is of considerable utility to be able to synthesize short (2-300 nucleotides) defined sequences of RNA, commonly referred to as RNA oligonucleotides or oligoribonucleotides. Over the past 25 years, many chemical approaches have been explored for synthesizing RNA oligonucleotides. Because deoxyribonucleic acid (DNA) methodologies have progressed more rapidly, the usual strategy for the synthesis of RNA has been to adapt DNA chemistries to RNA synthesis. Consequently, most approaches have focused on retaining the 5'-dimethoxytrityl (DMT) ether and adding a compatible 2'-hydroxyl protecting group such as fluoride-labile silyl ethers, photo-labile moieties, and acid-labile acetals. A delicate balance has been required to successfully utilize the acid-labile 2'-acetals in conjunction with the acid-labile 5'-DMT ether. Therefore, other approaches have involved retaining the 2'-acetal while replacing the 5'-DMT.
The acid-labile acetals have many attractive features. For example, it has been reported that it is possible to chromatograph some 2'-acetal-protected RNA. However, the subsequent removal of these acetals, which are used with the DMT group, requires acidic conditions which subsequently cause degradation of the RNA, or require extremely long periods of time to remove (&gt;24 hours). Therefore, although it may be possible to safely handle and purify some 2'-acetal-protected RNAs, the harsh conditions required for rapid deprotection may require further purification of the RNA, thereby negating this advantage. Milder conditions can be used but these are inconvenient, requiring more than 24 hours. More-labile acetals can not be used as they would not be sufficiently stable to the DMT acid-deprotection conditions during RNA synthesis.
Of all of the RNA synthesis methods reported to date, only the 5'-DMT-2'-t-butyldimethylsilyl (TBDMS) and the 5'-DMT-2'-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP) chemistries are readily available commercially. Unfortunately, neither of these methods allows RNA synthesis to be as routine and dependable as DNA synthesis. The impediments facing 5'-DMT-2'-FPMP chemistry are related to the problem of balancing two acid-labile protecting groups. One of the major difficulties with the 2'-TBDMS approach is that stepwise coupling yields are only 96-98% under routine conditions compared to &gt;99% for DNA. These methods enable the synthesis of RNA in acceptable yields and quality, but a high level of skill and significant investments in training and experience are required to deliver adequate results.
One of the most desirable conditions for the final 2'-deprotection of synthesized RNA is an extremely-mildly-acidic aqueous solution. In the optimal scheme, the 2'-protecting groups only have to be stable to withstand oligonucleotide synthesis conditions. Scaringe and Caruthers (U.S. patent application Ser. No. 08/488,878, filed Jun. 9, 1995 and incorporated herein by reference) recently reported a novel RNA synthesis strategy similar to such a scheme. Their investigations led to the development of silyl ethers for protection of the 5'-hydroxyl. However, this 5'-silyl ether oligonucleotide synthesis chemistry was not compatible with mildly-acid-labile 2'-acetals. Acid-labile orthoester protecting groups were investigated and discovered to have potential for use at the 2'-hydroxyl. The 2'-orthoesters that were developed in conjunction with 5'-silyl ethers enabled the synthesis of RNA oligonucleotides. Scaringe and Caruthers disclose specific orthoester protecting groups at the 2'-position of ribonucleotides. However, they do not disclose the orthoesters of the present invention.
The present invention provides an orthoester moiety that serves as a protecting group, particularly for RNA synthesis. Also provided in this invention is RNA comprising the protecting group which possesses novel advantages and useful features, e.g., a modified RNA oligonucleotide that is easily handled and analyzed with minimal concern about degradation. The protecting groups can be readily cleaved (&lt;10 minutes), if so desired, under extremely mild conditions that cause no detectable degradation of the RNA. No prior art anticipates that the following 2'-modification ##STR1## would be advantageous to RNA synthesis. This 2'-modification is the result of using an orthoester of the following general structure: ##STR2## where R represents protecting groups which can be removed prior to removing the orthoester.
The prior art has provided several means to synthesize RNA oligonucleotides. However, none have enabled the synthesis, handling, analysis and use of RNA oligonucleotides to be as robust and dependable as DNA synthesis, nor produced RNA comparable to the high quality in which DNA can be produced. No prior art has disclosed a modification of RNA that enables the RNA to be easily handled and then where the modification, e.g., a protecting group, can be removed under mild conditions to yield fully deprotected RNA. The present invention, therefore, provides more robust RNA synthesis methods which consistently produce higher quality RNA on a routine basis.